The calcium-dependent fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane, and the rapid retrieval of the vesicular membrane for reuse, underlies the efficiency and regulation of synaptic transmission. Although synaptic transmission is a specialized case of vesicular trafficking to the plasma membrane, it is representative of the cyclical process of vesicle trafficking that allows the placement and removal of proteins and lipids onto the plasma membrane. For example, high-affinity receptors are added to the plasma membrane to enable transduction of various signals and they are internalized for degradation or reuse, a process that alters their signaling. The control of receptor signaling is critical for many processes such as synaptic transmission, cell growth, and cellular differentiation. Cells require endocytosis not only for the uptake of essential nutrients from the extracellular environment but also to retrieve proteins and lipids that are added to the plasma membrane during fusion of regulated and constitutive secretory vesicles. Hrs-2 is a protein ATPase that has been implicated in vesicular trafficking by virtue of functional interactions with proteins known to be essential for both exocytosis and endocytosis. Our preliminary data suggest a principal role for hrs-2 in endocytosis because it is localized primarily on endosomes, has interactions with proteins critical for endocytosis, is homologous to a yeast protein required for endosomal protein trafficking, affects receptor mediated endocytosis, and inhibits endosome fusion. Understanding the role of hrs-2 in the molecular mechanisms of endocytosis is the subject of the proposed studies that are outlined in the following Specific Aims: 1. To characterize the interactions of hrs-2 with endocytic trafficking proteins. 2. To examine the intrinsic biochemical/biophysical properties of hrs-2. 3. To determine the cellular/molecular role of hrs-2 in specific steps of endocytic trafficking.